Homologous recombination allows numerous targeted genetic modifications in prokaryotic and selected eukaryotic organisms including selected deletions, insertions or replacements.
In higher eukaryotic organisms, homologous recombination may be stimulated through the induction of double stranded DNA breaks via rare-cutting endonucleases, such as e.g. I-SceI.
WO2004/067753 describes the use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and the application thereof for genome engineering and gene therapy.
WO2000/46386 describes methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell through I-SceI induced double stranded breaks. Also disclosed are methods of treating or phrophylaxis of a genetic disease in an individual in need thereof.
In plants, induction of double stranded DNA breaks using I-SceI has been shown to increase the frequency of homologous recombination by at least two orders of magnitude using Agrobacteria to deliver the repair DNA to the plant cells (Puchta et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp 5055-5060). Chilton and Que (2003, Plant Physiol. 133: pp 956-965) and Tzifira et al. (2003, Plant Physiol. 133: pp 1011-1023) report that T-DNA preferentially integrates in double stranded DNA breaks, artificially induced by the rare-cleaving enzymes I-SceI or I-CeuI. The reports also included donor T-DNA vectors which comprised a recognition site for the respective rare-cleaving enzyme.
In addition, methods have been described which allow the design of rare cleaving endonucleases to alter substrate or sequence-specificity of the enzymes, thus allowing to induce a double stranded break at virtually any locus of interest without being dependent on the presence of a recognition site for any of the natural rare-cleaving endonucleases. Briefly, chimeric restriction enzymes can be prepared using hybrids between a zinc-finger domain designed to recognize a specific nucleotide sequence and the non-specific DNA-cleavage domain from a natural restriction enzyme, such as FokI. Such methods have been described e.g. in WO 03/080809, WO94/18313 or WO95/09233 and in Isalan et al., 2001, Nature Biotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA 94, 5525-5530). Another way of producing custom-made meganucleases, by selection from a library of variants, is described in WO2004/067736. Custom made meganucleases with altered sequence specificity and DNA-binding affinity may also be obtained through rational design as described in WO2007/047859.
WO2007/049095 describes “LADGLIDADG” homing endonuclease variants having mutations in two separate subdomains, each binding a distinct part of a modified DNA target half site, such that the endonuclease variant is able to cleave a chimeric DNA target sequence comprising the nucleotides bound by each subdomain.
WO2007/049156 and WO 2007/093836 describe I-CreI homing endonuclease variants having novel cleavage specificity and uses thereof.
WO2007/047859 describes rationally designed meganucleases with altered sequence specificity and DNA binding affinity.
WO2006/105946 described a method for the exact exchange in plant cells and plants of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print and without resorting to in vitro culture during the removal step, employing the therein described method for the removal of a selected DNA by microspore specific expression of a double stranded break inducing rare cleaving endonuclease.
U.S. provisional patent application 60/828,042 and European patent application 06020370.0, and WO2008/037436 describe variants of the methods and means of WO2006/105946 wherein the removal step of a selected DNA fragment induced by a double stranded break inducing rare cleaving endonuclease is under control of a germline-specific promoter. Other embodiments of the method relied on non-homologous endjoining at one end of the repair DNA and homologous recombination at the other end.
Some of the embodiments of the above identified methods and means for exact exchange of a target DNA fragment for a DNA fragment of interest require that the introduced repair DNA is introduced in the plant cell in the presence of the double stranded break inducing enzyme. The repair DNA normally also contains the preselected site recognized by a double stranded break inducing rare cleaving endonuclease and therefore the repair DNA is also prone to DNA cleavage. Accordingly, the efficiency of DNA insertion by homologous recombination may be lowered. To avoid this decrease in efficiency, the preselected site in the repair DNA may be altered in such a way that it is no longer recognized by the double stranded break inducing rare cleaving endonuclease. However, this entails the introduction of an extra change in the repair DNA compared to the target DNA in addition to the desired change.
The current invention provides an alternative solution to this problem, which does not require the modification of the preselected site in the repair DNA and consequently allows the exchange of the target DNA with only the desired nucleotide change, without modification of the preselected site. These and other problems are solved as described hereinafter in the different detailed embodiments of the invention, as well as in the claims.